Comparative in silico characterization of Klebsiella pneumoniae hypervirulent plasmids and their antimicrobial resistance genes

Background The hypervirulent pathotype of Klebsiella pneumoniae (hvKp) is mainly mediated by large virulent plasmids. It seems that these hypervirulent plasmids (HVPs) are accumulating antimicrobial resistance genes (ARGs) and are turning quickly into drug-resistant hypervirulent hybrids. Therefore, molecular mechanisms involved in this convergence needs to be investigated to control their global spread. Methods In this study, the complete sequence of 79 non-redundant hypervirulent plasmids were retrieved from GenBank and their genetic features, hypervirulence and antimicrobial resistance patterns (AMR) as well as their putative transmission capability were compared using bioinformatics tools. Results The majority of HVPs belonged to clonal complex (CC)23, and sequence type (ST)11. IncFIB and IncHI1B were the most prevalent plasmid replicon types. Out of 79 plasmids, 78 were positive for iutA and iucA. The iucC, iucB and iucD genes were found in 77 plasmids. Almost 26% of the HVPs were potentially conjugative of which 71% carried AGRs. ARGs against beta-lactams, carbapenems, quinolones, aminoglycosides, chloramphenicols, tetracyclines and macrolides were detected in 30% of HVPs. Class 1 integron and prophage structures harboring multiple ARGs were found in eight plasmids. Insertion sequences (IS)6, IS110 and IS1380 appeared to be important genetic elements in transmission of ARGs. Conclusions The high prevalence of iucA and iutA suggests their strong capability for rapid and accurate genetic markers for discrimination of hvKp in the laboratory. This study indicated the important role of mobile genetic elements (MGEs) in the emergence of drug-resistance in hypervirulent strains. The high prevalence of putative conjugative hybrids implies higher incidence of multidrug-resistant (MDR)-hvKp strains in near future. Supplementary Information The online version contains supplementary material available at 10.1186/s12941-022-00514-6.


Introduction
Unlike classical Klebsiella pneumoniae as a major nosocomial opportunistic with high level of antimicrobial resistance (AMR); hypervirulent K. pneumoniae (hvKp) is a highly virulent pathogen and sensitive to antimicrobials [1]. It has the ability to cause community-acquired infections including liver abscesses, meningitis, endophthalmitis, necrotizing fasciitis, osteomyelitis as well as pneumonia [2]. The clinical outcome of hvKp are severe and mortality rate reaches 40% in blood-stream infections [3].
The hypervirulent pathotype of K. pneumoniae is attributed to the carriage of virulence genes involved in iron acquisition, hyper capsule production and heavy metal resistance [4,5]. This pathotype is mainly mediated by large hypervirulent plasmids (HVPs) [6,7]. Despite multiple investigations on molecular mechanisms

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Annals of Clinical Microbiology and Antimicrobials involved in hypervirulence of K. pneumoniae, a comprehensive study on extra-chromosomal hypervirulence reservoirs has not been conducted yet. The importance of HVPs increases when they encode antimicrobial resistance genes (ARGs) and turn into drug-resistant hypervirulent hybrids [8]. Previously, the prevalence of drug-resistant hvKp strains was low, however; reported cases have been increased worldwide and become a new threat [9]. Several studies from Europe, South America, Australia, and North America have reported MDR-hvKp [10]. Even though various ARGs have been detected in clinical hvKp strains, our knowledge on the role of HVPs in transmission of AMR genetic determinants among hypervirulent strains is still limited. The continuous accumulation of ARGs particularly in wide-spread sequence types (ST) of hvKp is troublesome. This is even more life-threatening in case of carpabememase-producers [11]. The mortality rate of carbapenemresistant hvKp sepsis has been reported as high as 100% [12]. In 2021, an extensively drug-resistant hvKp positive for bla NDM and bla OXA−48 was reported from a neonatal hospital [13]. Therefore, it is highly urgent to characterize AMR genetic reservoirs of HVPs to predict the future behavior of hvKp regarding antibiotic therapy.
In this analytical study, we characterized and compared the hypervirulence pattern of 79 pre-assembled HVPs of different K. pneumonia STs as well as their ARGs using bioinformatic tools. In addition, the plasmids were investigated for their replicon types, conjugation capability, plasticity and phylogenetic relationship.

Preparation of initial dataset
The term "hypervirulent K. pneumoniae" was searched in the GenBank database (https:// www. ncbi. nlm. nih. gov/ genba nk). The complete nucleotide sequence of all hypervirulent chromosomes and their plasmids were retrieved from GenBank.

Clonal relatedness of strains harboring HVPs
The distribution of HVPs among different hvKp STs were investigated. For each plasmid, the ST of its related chromosome based on seven housekeeping genes (gapA, infB, mdh, pgi, phoE, rpoB and tonB) was determined using the PubMLST database (https:// pubml st. org/) [14]. To characterize the clonal relationship of hvKp strains, the minimum spanning tree of all hvKp STs was prepared using PHYLOViZ version 2.0 [15].

Circular alignment and phylogenetic analysis of HVPs
To compare plasmids sequence plasticity, circular alignment was performed for several HVPs of major STs using the BLAST Ring Image Generator (BRIG) version 0.95 [16]. To investigate the phylogenetic relationship of the plasmids, a Neighbor Joining (NJ) tree was constructed based on genes with occurrence of > 10% in all plasmids using BacCompare (http:// bacco mpare. imst. nsysu. edu. tw) [17]. The cladogram was visualized using iTOL (https:// itol. embl. de) [18].

Genetic plasticity and phylogenic analysis and of HVPs
High heterogeneity was observed among different HVPs. However, they have similarity in some regions regardless of their strains STs. See Fig. 2A. A number of 2952 of genes with > 10% occurrence rate were identified for plasmids by BacCompare. The results showed that the HVPs had 1589 unique genes. The heatmap of 79 plasmids showed that the plasmids have different allele numbers in 185 loci. See Additional file 3: Fig. S1. The phylogenetic dendrogram based on 2952 gene classified the HVPs in three main clades (I, II and III). See Fig. 2B.

Genetic characteristics of HVPs
The size of plasmids ranged from 96,085 to 479,335 bp. Twenty-one plasmids were potentially conjugative carrying all four conjugal constituents including oriT, relaxase, type IV coupling protein (T4CP) and type IV secretion system (T4SS). Three plasmids including CP034421.1, CP045692.1 and CP032834.1 were mobilizable (only lacked oriT). All plasmids were positive for T4CP and T4SS.

Discussion
Up to now, various genetic markers have been dedicated to hvKP [27]. However, the definition of hvKP is still not fully established due to the complexity of virulence mechanisms [28]. The present study was focused on plasmid-mediated mediators of hypervirulence. We considered genes involved in iron acquisition, metabolite transport, tellurite resistance and hypermucoviscosity as markers of hypervirulence. However, hypermucoviscose has been considered a pathotype distinct from hypervirulent in some studies [29,30]. Moreover, the chromosomal carriage of genes such as entB, mrkD, fimH, wabG, ybtS, allS and kfu has been also investigated as hypervirulence mediators in several studies [3,8,30].
In consistent with other studies, CC23 was the most widely distributed CC among hvKp strains [31]. However, ST11 is the most prevalent clone in China [32]. In our study, 33% of hvKp strains belonged to ST11. Almost 41% of the HVPs were positive for all 14 hypervirulence genes of which 46% were isolated from CC23 and the remaining from ST11, ST286, ST375, ST86 and ST65. See Fig. 1. The hypervirulence patterns of strains belonging to a same ST were highly similar. In this regard, Yu et al. indicated that hvKp strains belonging to ST23, ST65, ST375 and ST86 were all positive for rmpA, rmpA2, iutA, and iroN on their plasmids.
Several studies have been conducted to find a method for accurate identification of hvKp in the laboratory [33]. The string test has been considered as a phenotypic marker for hvKp. However, its results are variable and sometimes unspecific [34]. Tellurite resistance was suggested as a screening method [33,35]. Hypervirulent populations were successfully separated based on their capsules using density-gradient centrifugation test [36]. Wu et al. distinguished hvKp based on positive string test and positive rmpA and iucA [37]. Molecular detection of rmpA and iucA was also used as hypervirulent indicator by Li et al. [3]. The peg-344, iroB, iucA, rmpA and rmpA2 genes showed > 95% diagnostic accuracy for identification of hvKp strains in 2018. The peg-344, iucA and iutA were introduced as the best markers for diagnosis of hvKp [38]. Based on the present virulence analysis, almost all HVPs harbored iucA and iutA. Therefore, molecular detection of iucA and iutA genes with accuracy of 98% can be advantageously used for precise screening of hvKp. The iucD, iucB, iucC, rmpA2, peg-589 and terB genes were also highly prevalent among HVPs (with the prevalence rate of 97%, 97%, 97%, 92%, 88% and 83%, respectively). This highlights their important role in hypervirulent pathotype of K. pneumoniae.
HvKp and classical K. pneumoniae have two distinct phenotypes with similar clonal lineages [27]. Their convergence can be mediated by the genetic integration of drugresistant plasmids in HVPs vice versa. Another probable mechanism is the transmission of ARGs in HVPs through recombinational processes. Also, it can result from the coexistence of antibiotic-resistant plasmids along with HVPs in one individual bacterium [39,40].
IncFIB was the most abundant (97%) replicon type among HVPs which co-existed with IncHI1B in 70% of the plasmids. The presence of specific Inc groups in majority of plasmids suggests that the formation of convergent phenotype is rarely result from plasmids integration whereas is more related to other two mentioned mechanisms. Also, it suggests that a drug-resistant plasmid with Inc groups other than IncFIB and IncHI1B has higher chance to reside in a single hvKp bacterium. were important due to the carriage of major ESBLs. Similar to classical plasmids, bla TEM and bla CTX-M were the most prevalent beta-lactamase producers among HVPs [41]. Almost 26% of the HVPs were potentially conjugative. See Fig. 2B. Interestingly, 71% of all putative conjugative HVPs carried AGRs. MK181633.1 was a conjugative MDR-HVP carrying the highest number of betalactamases and aminoglycosidases-producing genes. Of four carbapenem-resistant plasmids, three (MK181633.1, MK649825.1 and CP034201.1) harboring bla OXA-1 and bla NDM-1 were potentially conjugative. See Fig. 2B. Importantly, bla KPC-2 was carried by MF398271.1, a HVP harboring all 14 hypervirulent genes. Also, all quinolone resistance genes (qnrB4, qnrB17 and qnrS1) were carried by potentially conjugative HVPs. The isolation of selftransmissible HVPs carrying major ARGs suggests the increase of resistance against most clinically used antimicrobials by a few years.
Similar to classical plasmids, class 1 integron is highly responsible for resistance to multiple drug classes in hvKp strains [42]. Importantly, our data showed that resistance to third-generation cephalosporins and aminoglycosides is mediated by prophages structures in some hypervirulent strains. The extra-chromosomal phage-mediated carriage of ESBLs in hvKp has been previously reported by Yang et al. [43]. Moreover, IS6, IS110 and IS1380 seem to be important elements for horizontal transmission of major ARGs [44].
A specific distribution of antimicrobial-resistant HVPs was observed in the NJ dendrogram. The subclade distribution of plasmids within the leaves was not exactly associated with their strains STs. Conjugative antimicrobial-resistant HVPs were mainly distributed in clade II and III. Besides, almost all MDR-HVPs were located in clade III. While, clade I mainly contains non-drug-resistant HVPs. See Fig. 2B. Importantly, the results exhibited lower presence of hypervirulent genes in clade II and III. Based on our data, majority of highly antimicrobialresistant HVPs lacked iroN, iroD, iroC, iroB and peg344. By contrast, the full carriage of hypervirulent genes was mainly observed in plasmids belonging to clade I. This suggests that integration of antibiotic-resistant mediators in HVPs specifically occurs in plasmids with low levels of hypervirulence which are phylogenetically distinct from highly virulent plasmids to some extent.

Conclusion
We conducted a comprehensive molecular analysis on plasmids-borne mechanisms of hypervirulence. Based on the present analysis, molecular detection of the iucA and iutA genes can be advantageously utilized for accurate identification of hvKp. Moreover, we characterized the AMR genetic reservoirs of HVPs. Several ARGs against beta-lactams, carbapenems, quinolones, aminoglycosides, chloramphenicols, tetracyclines and macrolides were detected in HVPs. This study highlighted the role of MGEs in the emergence of AMR among hvKp strains. The presence of potentially conjugative MDR-HVP hybrids in this study implies higher incidence of drug-resistant hvKp strains in near future.